1. Field of the Invention
The present invention relates to processes for modifying proteins. More particularly, the present invention involves processes for linking polyethylene glycol to proteins in a manner which provides advantages associated with polyethylene glycol conjugated proteins while maintaining a desired protein bioactivity.
2. Description of Related Art
Processes and reagents for chemically modifying proteins have been used extensively for decades. Traditionally, protein chemical modifications were carried out in order to study their functional properties and structural characteristics. With the emergence of recombinant DNA techniques and interest in protein therapeutics, researchers have chemically modified proteins to improve their clinical performance. In particular, processes for conjugating proteins with polyethylene glycol have gained widespread use within the pharmaceutical and biochemical communities as a result of numerous improved pharmacological and biological properties associated with polyethylene glycol conjugated proteins. For example, polyethylene glycol conjugated proteins are known to have significantly enhanced plasma half life, and thus have substantially improved the clinical usefulness. Additionally, polyethylene glycol conjugated proteins generally have reduced antigenicity and immunogenicity, thereby are less prone to causing life-threatening anaphylaxis.
Another benefit associated with polyethylene glycol conjugated proteins is that of water solubility which is increased as a result of the high water solubility of polyethylene glycol. The increased water solubility can improve the protein""s formulation characteristics at physiological pH""s and can decrease complications associated with aggregation of low solubility proteins.
Additionally, polyethylene glycol conjugated proteins have found use in bioindustrial applications such as enzyme based reactions in which the reaction environment is not optimal for the enzyme""s activity. For example, some polyethylene glycol conjugated enzymes demonstrate a wider optimum pH activity and reduced optimum activity temperature. Moreover, enzymes having reduced activity in many organic solvents have been successfully conjugated with polyethylene glycol to a degree that renders them useful for catalyzing reactions in organic solvents. For example, polyethylene glycol has been conjugated with horseradish peroxidase which then becomes soluble and active in chloroform and toluene (Urrotigoity et al., Biocatalysis, 2:145-149, 1989).
Polyethylene glycol conjugated proteins vary in the extent to which plasma circulation half life is increased, immunogenicity is reduced, water solubility is enhanced, and enzymatic activity is improved. Factors responsible for these variations are numerous and include the degree to which the protein is substituted with polyethylene glycol, the chemistries used to attach the polyethylene glycol to the protein, and the locations of the polyethylene glycol sites on the protein.
The most common methods for attaching polyethylene glycol to proteins involve activating at least one of the hydroxyl groups on the polyethylene glycol with a functionality susceptible to nucleophilic attack by the nitrogen of amino groups on the protein. These methods generally result in loss of biological activity due to the nonspecific attachment of polyethylene glycol.
Alternative approaches to conjugating proteins with polyethylene glycol include controlling the conjugation reactants and conditions so that the conjugation site is confined to the N-terminus (Kinstler et al. Pharm. Res. 13:996, 1996); attaching polyethylene glycol to protein carbohydrate functionalities (Urrutigoity et al. Biocatalysis 2:145, 1989); attaching polyethylene glycol at protein cysteine residues (Goodson et al. Biotechnology 8:343, 1990); attaching polyethylene glycol during solid phase and solution phase peptide synthesis (Felix, ACS Symposium Series 680 ch 16, 1997) and, selectively replacing protein arginine residues with lysine residues that provide an polyethylene glycol attachment site (Hershfield et al. Proc. Natl. Acad. Sci. 88:7185, 1991). While these offer some degree of control of the reaction site, there is a continuing need for improved methods for providing polyethylene glycol conjugated proteins. In particular, it would be desirable to provide methods for conjugating proteins with polyethylene glycol that result in modified proteins having enhanced bioactivity or little loss in bioactivity while maintaining the benefits of polyethylene glycol conjugation, including substantially decreased immunogenicity, increased solubility, and prolonged circulation half lives characteristic of modified proteins.
The present invention provides protein modification processes that result in modified proteins having little or no decrease in an activity associated with the protein. More particularly, the invention described herein includes processes for modifying a protein by first deleting one or more amino acid sites on the protein that is suitable for polyethylene glycol conjugation and then contacting the protein with polyethylene glycol under conditions suitable for conjugating the polyethylene glycol to the protein. Preferably, the deleted amino acid residue is replaced with an amino acid residue that does not react with polyethylene glycol. The resulting polyethylene glycol conjugated protein has improved characteristics over proteins modified according to prior art procedures. An advantageous retention of activity is attributed to the availability of one or more protein binding sites which is unaltered in the protein modification process and thus remains free to interact with a binding partner subsequent to the modification process. Within the present invention are proteins useful for polyethylene glycol conjugation and polyethylene glycol conjugated proteins prepared by processes described herein.